The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

The paracellular channel for water secretion in the upper segment of the Malpighian Tubule of Rhodnius prolixus

Lumen to bath J ₁₂/C ₁ and bath to lumen J ₂₁/ C ₂ fluxes per unit concentration of 19 probes with diameters (d ^m) ranging from 3.0–30.0 Å (water, urea, erythritol, mannitol, sucrose, raffinose and 13 dextrans with d ^m 9.1–30.0 Å) were measured during volume secretion (J _v ) in the upper segment of the Malpighian Tubule of Rhodnius by perfusing lumen and bath with ¹⁴C or ³H-labeled probes. J _net=(J ₁₂/C ₁ – J ₂₁/C ₂) was studied as a function of J _v · J _v was varied by using different concentrations of 5-hydroxy tryptamine. J _net for ³H-water was not different from J _v We found: (i) A strong correlation between J _net and J _v for 8 probes d ^m =3.0–11.8 Å (group a probes), indicating that the convective component of J _net is more important than its diffusive component and than unstirred layers effects which are negligible. Therefore group a probes are solvent dragged as they cross the epithelium, (ii) There is no correlation between J _net and J _v for 11 probes with d ^m=11.8–30 Å (group b). Therefore these probes must cross the epithelium by diffusion and not by solvent drag, (iii) In a plot of J _net/J _v vs. d ^m group a probes show a steep linear relation with a slope = –0.111, while for group b probes the slope is –0.002. Thus there is a break between groups a and b in this plot. We tried to fit the data with models for restricted diffusion and convention through cylindrical or parallel slit pathways. We conclude that (i) group a probes are dragged by water through an 11.0 Å-wide slit, (ii) Most of J _v must follow an extracellular noncytosolic pathway, (iii) Group b probes must diffuse through a 42 Å-wide slit, (iv) A cylindrical pathway does not fit the data.E.G. is a Visiting Scientist at IVIC. It is a pleasure to thank Drs. A.E. Hill and Bruria Shachar-Hill for their suggestion of the use of dextrans, their instruction and help with the dextran separation technique, and their extensive discussions. Dr. R. Apitz, Mr H. Rojas and Mrs. Fulvia Bartoli were most helpful with suggestions during the course of the experimental work. Mr. Jose Mora was fundamental help with the equipment. Mrs. Lelis Ochoa and Mr. Luis F. Alvarez helped with some of the drawings. This work was partially supported by CONICIT, Fundación Polar and CDCH of UCV. It is a pleasure to thank Dr. H. Passow and Dr. K.J. Ullrich at the Max Planck Institut für Biophysik (Frankfurt/Main) where this work was initiated.     

Cross-flow ultrafiltration of proteins through asymmetric polysulfonic membranes: I. Retention curves and pore size distributions

Flux and retention of 0.1%w/w aqueous solutions of several proteins [lysozyme, pepsin, bovine serum albumin (BSA), lipase, and gamma-globulin] with molecular weights of 14.6, 36, 67, 801 and 150 kDa are studied when they are tangentially filtered, with transmembrane pressure differences until 1 MPa and circulation velocities in the re-tentate loop from 0.04 to 1.98 m/s (laminar regime), through two asymmetric polysulfone commercial membranes (E-100 with a nominal pore size of 0.01 mum and E-500 with a nominal pore size of 0.04 mum). Results are analyzed with the film theory for the concentration-polarization phenomenon, obtaining the mass transfer coefficient along with the apparent and true retention coefficients for the cell used, as a function of the feed circulation velocity and the molecular weight of the solute. The standard retention curves lead to pore size distributions differing from the nominal ones. These differences can be attributed to the modifications of the membranes when they are in operational conditions, probably due to protein adsorption. (c) 1995 John Wiley & Sons, Inc.     

Distribution and production ofChironomus salinarius (Diptera: Chironomidae) in a shallow coastal lagoon in the Bay of Cádiz

The abundance, generation time and production ofChironomus salinarius larvae in a lagoon fish-pond system in the Bay of Cádiz were studied by taking monthly samples at 3 sites during 1991 and 1992. Numerical abundance and biomass of larvae showed considerable spatial, seasonal and interannual variation (ANCOVAs,P<0.001). The maximum mean annual density was 7048 larvae m^–2, and corresponded to a biomass of 3.08 g dry weight (DW) m^–2. It was recorded at the site with the lowest rate of water renewal. Seasonal patterns were similar at all sites, with main annual peaks of abundance and biomass in autumn-early winter. Chironomid density was positively related to the biomass of benthic macroalgae (P<0.001). The population studied was multivoltine with a probable average of five generations per year, with overlapping cohorts and a predominance of third- and fourth-instar larvae. Estimates of annual production ranged between 72.2 g DW m^–2 yr^–1 at the site with the lowest rate of water renewal in 1991 and 0.1 g DW m^–2 yr^–1 at the site with the highest rate of water renewal in 1992. Mean annual production and the production/biomass ratio for the system was estimated to be 16.8 g DW m^–2 yr^–1 and 12.7, respectively. Possible factors leading to the observed density fluctuations are discussed, as well as possible sources of error in production estimates.     

Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD⁺ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD⁺-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD⁺ triggered Golgi disassembly by BFA. This effect of NAD⁺ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.     

Mast Cells Contain Large Quantities of Secretagogue-Sensitive N-Acetylaspartate

Abstract: Mast cells play a central role in both immediate allergic reactions and inflammation. A functional nerve-mast cell interaction has been proposed, given the morphological association between mast cells and neuropeptide-containing peripheral nerves. We now show that purified rat peritoneal mast cells contain large quantities of N -acetylaspartate (NAA; 747.50 nmol/mg of protein). Mast cell levels of NAA were rapidly reduced, by 64.0 and 86.4%, following treatment with compound 48/80 and mastoparan, respectively. These secretagogues strongly decreased mast cell histamine content over the same time period, suggesting also that NAA is stored in secretory granules. The data are the first to show that NAA is present in an immune effector cell type. Because NAA may be involved in myelin synthesis and glutamyl peptide metabolism, NAA released from mast cells following nervous or other stimuli could participate in neuroimmune interactions. Mast cells in multiple sclerosis plaques may contribute to the reported elevations in brain NAA in this disease.     

A simple mathematical model of the interaction between intracranial pressure and cerebral hemodynamics

Ursino, Mauro, and Carlo Alberto Lodi. A simplemathematical model of the interaction between intracranial pressure andcerebral hemodynamics. J. Appl.Physiol. 82(4): 1256-1269, 1997.A simplemathematical model of intracranial pressure (ICP) dynamics oriented toclinical practice is presented. It includes the hemodynamics of thearterial-arteriolar cerebrovascular bed, cerebrospinal fluid (CSF)production and reabsorption processes, the nonlinear pressure-volumerelationship of the craniospinal compartment, and a Starling resistormechanism for the cerebral veins. Moreover, arterioles are controlledby cerebral autoregulation mechanisms, which are simulated by means ofa time constant and a sigmoidal static characteristic. The model isused to simulate interactions between ICP, cerebral blood volume, andautoregulation. Three different related phenomena are analyzed: thegeneration of plateau waves, the effect of acute arterial hypotensionon ICP, and the role of cerebral hemodynamics during pressure-volume index (PVI) tests. Simulation results suggest the following:1) ICP dynamics may become unstablein patients with elevated CSF outflow resistance and decreasedintracranial compliance, provided cerebral autoregulation is efficient.Instability manifests itself with the occurrence of self-sustainedplateau waves. 2) Moderate acutearterial hypotension may have completely different effects on ICP,depending on the value of model parameters. If physiological compensatory mechanisms (CSF circulation and intracranial storage capacity) are efficient, acute hypotension has only negligible effectson ICP and cerebral blood flow (CBF). If these compensatory mechanismsare poor, even modest hypotension may induce a large transient increasein ICP and a significant transient reduction in CBF, with risks ofsecondary brain damage. 3) The ICPresponse to a bolus injection (PVI test) is sharply affected, viacerebral blood volume changes, by cerebral hemodynamics andautoregulation. We suggest that PVI tests may be used to extractinformation not only on intracranial compliance and CSF circulation,but also on the status of mechanisms controlling CBF.

     

Production of neosolaniol byFusarium tumidum

Extracts from autoclaved maize culture ofFusarium tumidum strain R-5823 were toxic towardsArtemia salina. Bioassay-guided fractionation of the organic extract led to the isolation of the toxic compound that was identified as the trichothecene toxin neosolaniol (NEOS) by¹H,¹³C nuclear magnetic resonance spectroscopy and low-resolution electronic impact mass spectrometry. The amount of NEOS produced by the strain R-5823 was 300 mg/kg maize culture. NEOS was also detected by HPLC in cultures of four out of seven additional strains ofF. tumidum andGibberella tumida with different origin, in amounts ranging from 1 to 311 mg/kg. This is the first report on the production of a trichothecene toxin byF. tumidum.     

Mauthner cell-initiated abrupt increase of the electric organ discharge in the weakly electric fishGymnotus carapo

Stimulation of the spinal cord of the electric fish Gymnotus carapo, evoked an abrupt increase in the discharge rate of the electric organ. At the maximum of this response, the rate increased an average of 26 ± 11.8%. The duration of the response was 4.9 ± 2.12 s; its latency was 10.4 ± 1.1 ms. Activation of the Mauthner axon played a decisive role in this phenomenon as indicated by the following: (1) recordings from the axon cap of the Mauthner cell demonstrated that the response was evoked if the Mauthner axon was antidromically activated and (2) a response that was similar to that produced by spinal cord stimulation, was elicited by intracellular stimulation of either Mauthner cell. Stimulation of the eighth nerve could also increase the discharge rate of the electric organ. The effect was greater if a Mauthner cell action potential was elicited. The findings described in the present report, indicate the existence of a functional connection between the Mauthner cell and the electromotor system in Gymnotus carapo. This connection may function to enhance the electrolocative sampling of the environment during Mauthner-cell mediated behaviors. This is a novel function for the Mauthner cell.Abbreviations EHP extrinsic hyperpolarizing potential - EOD electric organ discharge - M-AIR Mauthner initiated abrupt increase in rate - M-cell Mauthner cell - M-axon Mauthner axon - PM pacemaker nucleus - PM-cell pacemaker cell - PPn prepacemaker nucleus - SPPn sublemniscal prepacemaker nucleus